Herpes simplex virus (HSV) is the leading infectious agent causing blindness world wide. This human viral pathogen invades the nervous system, seeding host neurons with viral genetic information. This genetic information is a stable repository from which the virus reinitiates lytic infection which can result in recurrent or persistent corneal inflammation, scarring and ultimately blindness. The molecular mechanisms underlying this process are not well understood but remain central to the prevention and control of HSV. The long-term goal of the proposed research is to define the molecular mechanisms involved in the establishment of herpes simplex virus (HSV) latency. Using a novel single cell PCR assay to obtain quantitative information about the number of neurons containing the viral genome, we have demonstrated that a 2.3kb fragment residing within the latency associated transcript (LAT) gene provides a critical function for the establishment of latent infections (Thompson and Sawtell J.Virol.71 :5432, 1997). Data has been recently generated indicating a significant increase in neuronal death in ganglia infected with mutants lacking this 2.3kb fragment. Consistent with the decreased neuronal survival in the peripheral nervous system, LAT null mutants are more neuroinvasive. Pfu/LD50 experiments demonstrate LAT null mutants in strain l7syn+ are 100-fold more virulent when compared to wild type or genomically rescued isolates. Our ability to precisely quantify the number of latently infected neurons in the ganglia and the number of viral genomes in those individual neurons will be exploited to evaluate genetically engineered mutant strains to define the regulatory mechanisms by which locus attenuates virulence increases the latent repository of viral genetic information, and promotes reactivation and thereby recurrent disease. The information gained in these studies will permit the design of more effective vaccines and interventive drug therapies.